This pilot project proposes to develop a means by which pluripotent stem cells transduced with the fetal hemoglobin gene can be chemically selected in vivo. We propose that expression in the stem cells of an appropriate human glutathione S-transferase (GST) will confer resistance to the alkylating agent busulfan and allow for stem cell selection. The method proposed takes advantage of the very high susceptibility of stem cells to the alkylating agent busulfan, the importance of glutathione metabolism in terminating the activity of busulfan in vivo, the ability of transfection of cells with glutathione S-transferase to confer resistance to alkylating agents, and the correlation of resistance to hepsulfam (a close chemical relative of busulfan) with GST expression in human breast cancer cell lines. We will identify, through isolation of human GST from liver and placenta and confirmation with E. coli expressed enzyme, the GST most active in busulfan-glutathione conjugation. The investigators in Project I will introduce the corresponding gene together with a LCR gamma globin gene into hematopoietic stem cells, and we will together evaluate the effectiveness of this strategy by assaying resistance of the transduced cells and their ability to form a busulfan glutathione conjugate in comparison to wild type cells.